To date, most traditional vaccines have been composed of live-attenuated or inactivated whole pathogen preparations. Generation of these sorts of vaccines is limited by the requirement for long and intensive basic research and development. Reliable production and scale-up technologies for live-attenuated or inactivated vaccines would be almost impossible to develop at short notice. There is, therefore, a need for the development of a safe, robust and broadly-useful technology that is suitable for the production of vaccines against unanticipated infectious disease threats. Vaccines developed from plant-virus-pathogen chimera's may provide a method to rapidly produce vaccines that can be used to prevent or treat a number of known or emerging disease threats.
Controlling immune responses to pathogens and tumor cells has been the focus of immunology, cell biology and pharmaceutical development for several decades. Much has been learned about the complexity of immune cells and the patterns and effect of cytokine expression in response to pathogen challenge, and vaccine administration. One key aspect of this work has been the identification of two major arms of the immune response, the Th1 response, which is largely cellular, and the Th2 response, which is predominantly humoral. The two types of immune responses are mounted in response to how foreign antigens are presented to the immune system, what cytokines are expressed by presenting cells and what types of immune cells are activated. Th1 responses result in cytotoxic immune cell function and production of neutralizing antibodies of a different subtype than observed with Th2 responses. While some pathogens can be susceptible to Th2 responses, the Th1 response is key to mounting an effective response to both pathogen and tumor cells. However, both pathogens and tumor cells have developed strategies to avoid immune surveillance, bypassing mechanisms that are essential to Th1 immunity.
A key goal in vaccine development is to direct Th1 type immunity, in addition to Th2 humoral responses, upon vaccine administration to the host. By using an attenuated cowpox virus, Jenner unknowingly took advantage of the powerful activation of Th1 pathway to prevent smallpox infections. Since his time, most pathogen vaccines have been killed or attenuated, which have generally shown good success in controlling pathogen morbidity and viral spread. However, two aspects of recent vaccine development have led to growing concerns for live or attenuated viral vaccines. The use of an attenuated or killed virus to treat human immunodeficiency virus (HIV) is impractical for several reasons. Occupational safety concerns, low yield of attenuated virus, and the threat of viral mutation or escape are serious drawback to both vaccine development and public acceptance. In other cases, as observed with measles virus and respiratory syncytial virus (RSV), unpredictable and severe adverse events are associated with whole virus immunization. Therefore, much research has focused on “subunit” vaccines, which are composed of pathogen protein(s) or peptides that are generally targeted by the host immune response for protective immunity (Vaccines, 3rd ed 1999, Plotkin and Orenstein, Philadelphia Pa., Saunders Co). Unfortunately, protein subunit vaccines don't often elicit strong Th1 responses by themselves, and DNA subunit vaccines often fail to elicit antibodies. In most cases both antibodies and CTL responses are necessary in controlling pathogenesis or disease progression.
Two new types of vaccines have been created to overcome the deficiencies of current subunit vaccines. Non-pathogenic viruses have been genetically modified to encode immunogenic subunit proteins of a pathogen, thus taking advantage of the Th1 immune response to viral antigen presentation. Strong Th1 type immune responses have been demonstrated for many pathogen and self-antigens using adenovirus, vaccinia, fowlpox and alphavirus delivery systems (Walther and Stein. 2000 Drugs 60, 249). However, these “first generation” viral delivery systems encountered problems due to the vector immunogenicity, which precluded their subsequent use in booster immunizations. Viral priming followed by either protein or DNA boosting has been successful, but this approach requires the manufacture of at least two agents for a single vaccine. The large-scale manufacture of DNA and/or protein for these vaccines has encountered both technical and financial challenges.
A second strategy takes advantage of the self-assembly of viral coat proteins into virus like particles (VLPs), which by themselves stimulate strong Th1 antigen responses (Schiller and Lowy. 2001 Expert Opin Biol Ther. 1, 571). VLPs constructed from arrayed viral coat have been shown to be effective in stimulating both neutralizing antibody and cytotoxic T lymphocyte (CTL) responses. Viral coat proteins are also effective carriers of antigens through fusion to the external solvent-exposed residues, usually by genetic fusion (Pogue et al. 2002 Ann Rev Phyto Path 40, 3; Da Silva. 1999 Curr Opin Mol Ther 1, 82). Though promising, VLP technology also has drawbacks. Production is again limiting, and often fusion of a heterologous antigen to the coat reduces VLP yield, solubility, or prevents self-assembly. In addition, immune clearance, the same mechanism that limits whole virus boosting, also limits the use of VLPs. Clearly, there is a need for a cost effective viral coat antigen delivery system that overcomes the limitations of both whole virus and VLP technology for vaccine delivery. The properties of this system would include all the benefits of boosting Th1 responses via a virus-like antigen presentation to the immune system without pathogenicity, flexibility to rotate the VLP backbone to which the antigen is fused, generation of and control of immunogenicity, high yield and low cost.
Applicant and others have shown that coat proteins from plant viruses have all the immunologic presentation properties of mammalian virus coat, but without pathogenicity. A large number of positive (+) strand RNA plant viruses, including Tobacco Mosaic Virus (TMV), type member of the tobamovirus family, have been cloned and manipulated in vitro to express heterologous gene products in plants as well as to display biologically relevant peptides on its virion surface. A unique property of TMV virions is their ability to be disassociated to form monomers and self assemble into VLPs using a RNA scaffold. Plant coat proteins, including TMV, engineered to display foreign epitopes have been shown to promote functional immunity to both self-antigens (Savelyeva N 2001 Nat Biotechnol 19 760) and various pathogens (Pogue et al. 2002 Ann Rev Phyto Path 40, 3).
Essential for the encapsidation of the viral genomic RNA molecule into an infectious particle is the presence of a sequence element referred to as the origin of assembly (OAS). The TMV OAS is located approximately 1 Kb from the 3′ end of the viral genome and consists of a 440 nucleotide sequence that is predicted to form three hairpin stem-loop structures (Turner and Butler, 1986). The viral coat protein disks initially bind to loop 1 during viral assembly. In vitro packaging assays using mutual assembly origin transcripts have defined the 75 nucleotides comprising loop 1 as necessary and sufficient for encapsidation of foreign or viral RNA sequences (Turner et al., 1988). In vitro reconstitution studies have shown that preparations of purified coat protein, derived from virions from infected plant cells, are able to assemble into helical structures with TMV RNA at pH 7.0, resulting in assembly of TMV-like viral particles containing RNA (Fraenkel-Conrat and Williams, 1955). Furthermore, it has been shown that foreign chimeric RNA molecules containing OAS sequences, transcribed in vitro using SP6 or T7 RNA polymerase, may be assembled in vitro into pseudovirus particles (Sleat et al., 1986).
The cloning and sequencing of the viral coat proteins responsible for encapsidation has led to the insertion of these genes into bacterial expression vectors in, for example, E. coli (Shire et al., 1990). However, in vitro assembly with recombinant E. coli viral coat proteins results in a decreased reconstitution rate relative to native coat protein produced in plants (Shire et al., 1990). U.S. Pat. No. 5,443,969 attempts to overcome this deficiency in E. coli by packaging RNA sequences containing a TMV-OAS in vivo in E. coli, instead of in vitro. However, introduction of the encapsidated viral vectors into hosts outside of plants is problematic. The lack of acetylation of the TMV coat protein in E. coli results in poorly efficient encapsidation of non-capped RNAs. These RNAs are poorly translated in eukaryotic cells due to the lack of the cap structure. Further, the yields of recombinant TMV products in E. coli are very poor and not commercially feasible.
The process of intracellular delivery of genetic material for therapeutic purposes by either correcting an existing abnormality or providing cells with a new function is the basis behind gene therapy (Drew and Martin, 1999), and for DNA immunizations. Practically speaking, nucleic acid immunization technologies present an attractive front-line defense against new pathogens: there is probably no other system that can compete as the first line in a rapid-response subunit vaccine strategy. However, conventional DNA vaccines suffer from a number of significant drawbacks that makes reliance on this technology alone unwise. Most significantly, the dose of DNA required to stimulate an effective immune responses is very high, with the implication that production of significant quantities for large scale immunization will be challenging. DNA and RNA vaccines are generally capable of promoting good Th1 type cytotoxic T cell responses, which are essential for elimination of non-cytopathic pathogens. However, with few exceptions, the antibody response induced by DNA vaccines is poor. Hence, although nucleic acid vaccines are attractive from the prospective that production can be very rapid, ideally an initial DNA or RNA vaccination should be followed by a booster vaccination, preferably with protein, to induce efficient antibody production and more complete protection against pathogen challenge. The current invention addresses the issues raised above by introducing a novel and flexible vaccine delivery platform